This invention relates to methods and compositions for forming and using sustained release microspheres. The microspheres are porous and include a plurality of channel openings that are less than 1000 angstroms in diameter.
Microparticles, microspheres, and microcapsules, referred to herein collectively as xe2x80x9cmicroparticlesxe2x80x9d, are solid or semi-solid particles having a diameter of less than one millimeter, more preferably less than 100 microns, which can be formed of a variety of materials, including synthetic polymers, proteins, and polysaccharides. Microparticles have been used in many different applications, primarily separations, diagnostics, and drug delivery.
The most well known examples of microparticles used in separations techniques are those which are formed of polymers of either synthetic or protein origin, such as polyacrylamide, hydroxyapatite or agarose. These polymeric microparticles are commonly used to separate molecules such as proteins based on molecular weight and/or ionic charge or by interaction with molecules chemically coupled to the microparticles.
In the diagnostic area, microparticles are frequently used to immobilize an enzyme, substrate for an enzyme, or labeled antibody, which is then interacted with a molecule to be detected, either directly or indirectly.
In the controlled drug delivery area, molecules are encapsulated within microparticles or incorporated into a monolithic matrix, for subsequent release. A number of different techniques are routinely used to make these microparticles from synthetic polymers, natural polymers, proteins and polysaccharides, including phase separation, solvent evaporation, emulsification, and spray drying. Generally the polymers form the supporting structure of these microspheres, and the drug of interest is incorporated into the polymer structure. Exemplary polymers used for the formation of microspheres include homopolymers and copolymers of lactic acid and glycolic acid (PLGA) as described in U.S. Pat. No. 5,213,812 to Ruiz, U.S. Pat. No. 5,417,986 to Reid et al., U.S. Pat. No. 4,530,840 to Tice et al., U.S. Pat. No. 4,897,268 to Tice et al., U.S. Pat. No. 5,075,109 to Tice et al., U.S. Pat. No. 5,102,872 to Singh et al., U.S. Pat. No. 5,384,133 to Boyes et al., U.S. Pat. No. 5,360,610 to Tice et al., and European Patent Application Publication Number 248,531 to Southern Research Institute; block copolymers such as tetronic 908 and poloxamer 407 as described in U.S. Pat. No. 4,904,479 to Illum; and polyphosphazenes as described in U.S. Pat. No. 5,149,543 to Cohen et al. Microspheres produced using polymers such as these exhibit a poor loading efficiency and are often only able to incorporate a small percentage of the drug of interest into the polymer structure. Therefore, substantial quantities of microspheres often must be administered to achieve a therapeutic effect.
Spherical beads or particles have been commercially available as a tool for biochemists for many years. For example, antibodies conjugated to beads create relatively large particles specific for particular ligands. The large antibody-coated particles are routinely used to crosslink receptors on the surface of a cell for cellular activation, are bound to a solid phase for immunoaffinity purification, and may be used to deliver a therapeutic agent that is slowly released over time, using tissue or tumor-specific antibodies conjugated to the particles to target the agent to the desired site.
The most common method of covalently binding an antibody to a solid phase matrix is to derivative a bead with a chemical conjugation agent and then bind the antibody to the activated bead. The use of a synthetic polymeric bead rather than a protein molecule allows the use of much harsher derivatization conditions than many proteins can sustain, is relatively inexpensive, and often yields a linkage that is stable to a wide range of denaturing conditions. A number of derivatized beads are commercially available, all with various constituents and sizes. Beads formed from synthetic polymers such as polyacrylamide, polyacrylate, polystyrene, or latex are commercially available from numerous sources such as Bio-Rad Laboratories (Richmond, Calif.) and LKB Produkter (Stockholm, Sweden). Beads formed from natural macromolecules and particles such as agarose, crosslinked agarose, globulin, deoxyribose nucleic acid, and liposomes are commercially available from sources such as Bio-Rad Laboratories, Pharmacia (Piscataway, N.J.), and IBF (France). Beads formed from copolymers of polyacrylamide and agarose are commercially available from sources such as IBF and Pharmacia. Magnetic beads are commercially available from sources such as Dynal Inc. (Great Neck, N.Y.).
One disadvantage of the microparticles or beads currently available is that they are difficult and expensive to produce. Microparticles produced by these known methods have a wide particle size distribution, often lack uniformity, and fail to exhibit long term release kinetics when the concentration of active ingredients is high. Furthermore, the polymers used in these known methods are dissolved in organic solvents in order to form microspheres. The microspheres must therefore be produced in special facilities designed to handle organic solvents. These organic solvents could denature proteins or peptides contained in the microparticles. Residual organic solvents could be toxic when administered to humans or animals.
In addition, the available microparticles are rarely of a size sufficiently small to fit through the aperture of the size of needle commonly used to administer therapeutics or to be useful for administration by inhalation. For example, microparticles prepared using polylactic glycolic acid (PLGA) are large and have a tendency to aggregate. A size selection step, resulting in product loss, is necessary to remove particles too large for injection. PLGA particles that are of a suitable size for injection must be administered through a large gauge needle to accommodate the large particle size, often causing discomfort for the patient.
Generally all currently available microspheres are activated to release their contents in aqueous media and therefore must be lyophilized to prevent premature release. In addition, particles such as those prepared using the PLGA system exhibit release kinetics based on both erosion and diffusion. In this type of system, an initial burst or rapid release of drug is observed. This burst effect can result in unwanted side effects in patients to whom the particles have been administered.
Microparticles prepared using lipids to encapsulate target drugs are currently available. For example, lipids arranged in bilayer membranes surrounding multiple aqueous compartments to form particles may be used to encapsulate water soluble drugs for subsequent delivery as described in U.S. Pat. No. 5,422,120 to Sinil Kim. These particles are generally greater than 10 xcexcm in size and are designed for intra articular, intrathecal, subcutaneous and epidural administration. Alternatively, liposomes have been used for intravenous delivery of small molecules. Liposomes are spherical particles composed of a single or multiple phospholipid and cholesterol bilayers. Liposomes are 30 xcexcm or greater in size and may carry a variety of water-soluble or lipid-soluble drugs. Liposome technology has been hindered by problems including purity of lipid components, possible toxicity, vesicle heterogeneity and stability, excessive uptake and manufacturing or shelf-life difficulties.
Therefore, there is an on-going need for development of new methods for making microparticles, particularly those that can be adapted for use in the separations, diagnostic and drug delivery area. Preferably, such improved microparticles would permit the sustained release of active agents in a predictable, consistent manner.
The invention solves these and other problems by providing methods and compositions for the sustained release of therapeutic and/or diagnostic agents in vivo and in vitro. As subsequently used herein the term xe2x80x9ctherapeutic agentxe2x80x9d is intended to be inclusive of clinical agents which can be administered in microcapsular form, whether used primarily for treatment or diagnosis.
According to one aspect of the invention, a microsphere having a smooth surface which includes a plurality of channel openings is provided. The channel openings have a diameter which is less than 1000 angstroms as determined by gas adsorption technique for pore sizing. The microspheres of the invention include a macromolecule, preferably a protein or a nucleic acid, and at least one water soluble polymer. In general, the microspheres are formed by contacting the macromolecule and at least one water soluble polymer under aqueous conditions to form the microspheres, and the microspheres are then stabilized by either chemical crosslinking or exposing the microspheres to an energy source, preferably heat, or both, at a temperature which results in microspheres which are resistant to physical and chemical treatments such as sonication and caustic solutions under these conditions. Although not wishing to be bound to any particular theory or mechanism, it is believed that the microspheres form during the mixing step; however, such initially formed microspheres are transient and require a further step (e.g., including a crosslinking agent in the mixture and/or by applying heat or other energy source) to stabilize the transiently-formed microspheres. The particular conditions for forming representative microspheres of the invention are described in the Examples. In the preferred embodiments, the formation reaction is conducted in the absence of the addition of oils or organic solvents. Oil is defined as a substance that is liquid fat which is insoluble in water.
The protein component of the microsphere may be a carrier protein or a therapeutic protein (see, e.g., Table 1). As used herein, a xe2x80x9ccarrier proteinxe2x80x9d refers to a protein which has a molecular weight of at least about 1500 and which exists as a three dimensional structure. The carrier protein can also be a therapeutic protein, i.e., a protein which has a therapeutic activity; however, in general, the phrase xe2x80x9ccarrier proteinxe2x80x9d will be used in this application to refer to a protein which has a primary function to provide a three dimensional structure for the purpose of microsphere formation, even if the carrier protein also may have a secondary function as a therapeutic agent. In certain preferred embodiments, the carrier protein is an albumin, particularly, human serum albumin. The protein microspheres of the invention, optionally, further include a therapeutic agent such as a steroid (e.g., estradiol, testosterone, prednisolone, dexamethasone, hydrocortisone, lidocaine base, procaine base), or any other such chemical entity known to bind to the protein, preferably albumin, such as GCSF, or paclitaxel. In yet other embodiments (discussed below), the microspheres of the invention further include a complexing agent and, more preferably, a therapeutic agent (preferably a peptide) which is associated with the complexing agent via an ionic or nonionic interaction. In certain other embodiments, the protein that comprises the matrix is a therapeutic protein (e.g., a hormone such as insulin or human growth hormone) and the microsphere is constructed and arranged to provide sustained release of the therapeutic protein in vivo. More preferably, the microsphere is constructed and arranged to provide sustained release of the therapeutic agent in the absence of significant swelling of the microsphere.
Surprisingly, the surface of the microsphere is different from the interior. Extensive water washing of freeze fractured microspheres dissolves much of the microsphere matrix material leaving a thin shell. In addition, the surface of the microsphere is smooth; the channel (pore) openings are less than 1000 angstroms in diameter as determined by gas adsorption technique for por sizing using, e.g., BET technology for data analysis.
In general, the microspheres of the invention are formed by mixing the macromolecule, preferably a protein or a nucleic acid, together with at least one water polymer under conditions which permit the water soluble polymer to remove water from (xe2x80x9cdehydratexe2x80x9d) the macromolecule within specified or preferred ratios of macromolecule to water soluble polymer. As used herein, a xe2x80x9cwater soluble polymerxe2x80x9d of the invention refers to a polymer or mixture of polymers which is capable of removing water from or dehydrating the macromolecule or otherwise capable of causing volume exclusion. Thus, the preferred process involves volume exclusion using an entirely aqueous system with no oil or organic solvents involved.
Suitable water soluble polymers include soluble linear or branched polymers, preferably those having a high molecular weight. Polymers can be highly water soluble, moderately-water soluble, or slightly water soluble (greater than 2% wt/vol water soluble). The preferred water soluble polymers are water soluble or soluble in a water miscible solvent. The water soluble polymers may be solubilized by first being dissolved in a water miscible solvent and then combining the polymer solution with an aqueous solvent. In the particularly preferred embodiments, the water soluble polymers of the invention are selected from the water soluble polymers identified in Table 2. In certain embodiments, the microspheres of the invention are formed of proteins and water soluble polymers and contain from 40 to less than 100 (wt %) protein. The final microsphere product which has been stabilized using a crosslinking agent and/or exposure to an energy source such as heat does not swell significantly in the presence of water (i.e., it is not a hydrogel). In the particularly preferred embodiments, the water soluble polymer is a carbohydrate-based polymer. Thus, in certain preferred embodiments, the microsphere comprises: (1) a protein, preferably albumin; and (2) a carbohydrate-based water soluble polymer, preferably hetastarch, wherein the protein represents at least 40% and less than 100% by weight of the microsphere. Preferably, the carbohydrate-based polymer represents greater than 0% and less than or equal to 30% by weight of the microsphere. In these and other embodiments, the microspheres preferably further comprise an active agent, preferably a luteinizing hormone releasing hormone or analog thereof. In general, the microspheres of the invention, when contacted with a solution of active agent, are capable of incorporating at least 60%, more preferably at least 70%, at least 80%, or at least 90%, and most preferably, at least 95% or at least 98% of the active agent. The active agent-containing microspheres optionally are further stabilized by contacting the microspheres with the same types of crosslinking agents and using the same types of conditions described herein for initially stabilizing the microspheres.
According to another aspect of the invention, a microsphere further including a complexing agent is provided. The microsphere of this aspect includes: (1) a macromolecule such as a protein (e.g., albumin, as described above); (2) at least one water soluble polymer (e.g., hetastarch (hydroxyethylstarch), PEG/PVP); and (3) a complexing agent. As used herein, a complexing agent refers to a molecule which is capable of interacting with a therapeutic agent (discussed below) to facilitate loading, retaining and/or otherwise delaying the release of the therapeutic agent from the microsphere (see, e.g., Table 3). As with all aspects of the invention, these microspheres have a smooth surface which includes a plurality of channel openings that are less than 1000 angstroms in diameter as determined by gas adsorption technique for pore sizing and, preferably, do not contain detectable oil or organic solvent.
According to a particularly preferred aspect of the invention, a microsphere further including at least two complexing agents is provided. The microsphere of this aspect includes: (1) a macromolecule such as a carrier protein (e.g., albumin, as described above); (2) at least one water soluble polymer (e.g., hetastarch (hydroxyethylstarch), PEG/PVP); (3) a first complexing agent that is an anionic polysaccharide; and (4) a second complexing agent that is a divalent metal cation selected from the group consisting of calcium, magnesium, zinc, strontium, barium, manganese, and iron. A particularly preferred embodiment of this aspect of the invention is illustrated in Example 17. Calcium and magnesium are the preferred divalent metal cations.
As used herein, a complexing agent refers to a molecule which is capable of interacting with a therapeutic agent (discussed below) to facilitate loading, retaining and/or otherwise delaying the release of the therapeutic agent from the microsphere (see, e.g., Table 3). As with other aspects of the invention, these microspheres preferably have a smooth surface which includes a plurality of channel openings that are less than 1000 angstroms in diameter as determined by gas adsorption technique for pore sizing and, more preferably, do not contain detectable oil or organic solvent.
A preferred method of incorporating the complexing agent(s) is to combine the complexing agent(s) with a water soluble polymer in aqueous solution, then add the macromolecule, and stabilize the microspheres with heat and/or crosslinking agents.
In general, the complexing agent is an ionic complexing agent (i.e., the complexing agent is capable of an ionic interaction with a therapeutic agent (discussed below)) or a non-ionic complexing agent (i.e., the complexing agent is capable of a non-ionic (e.g., hydrophobic, mixed ionic/nonionic) interaction with a therapeutic agent or with another complexing agent). Exemplary complexing agents are provided in Table 3. Ionic complexing agents of the invention are further categorized into anionic complexing agents (i.e., complexing agents having a negative charge such as anionic polysaccharides, e.g., dextran sulfate, galacturonic acids, alginates, mannuronic acid, guluronic acid, hyaluronic acid, chondroitin sulfates, heparin, chitin, chitosan, glycosaminoglycans, proteoglycans) and cationic complexing agents (i.e., complexing agents having a positive charge). The preferred complexing agents of the invention are anionic polysaccharides and divalent metal cations selected from the group consisting of calcium and magnesium.
In certain embodiments, the complexing agent is an anionic complexing agent having the structure of formula I:
POLY-[Yxe2x88x92]n X+xe2x80x83xe2x80x83I.
wherein POLY represents a principal chain of the anionic complexing agent which may be linear or branched;
wherein Yxe2x88x92 represents an anionic group, e.g., sulfates, carboxyls, phosphates, nitrates, carbonates and the like, that may be coupled to any one or more of the branches of the principal chain;
wherein X+ represents a cationic group, e.g., that is a counter ion to the anionic group;
wherein n is an integer from 1 to 10,000, preferably, from 5 to 100 and, more preferably, from 5 to 1000, and still more preferably, from 5 to 10,000; and
wherein when n is greater than 1, the n Yxe2x88x92 groups can be the same or different.
In yet other embodiments of the invention the complexing agent is a cationic complexing agent having the structure of formula II:
POLY-[X+]n Yxe2x88x92xe2x80x83xe2x80x83II.
wherein POLY represents a principal chain of the cationic complexing agent which may be linear or branched;
wherein X+ represents a cationic group, e.g., an amino group, that may be coupled to any one or more of the branches of the principal chain;
wherein Yxe2x88x92 represents an anionic group, that is a counter ion to the cationic group;
wherein n is an integer from 1 to 10000, preferably, from 5 to 100 and, more preferably, from 5 to 1000, and most preferably, from 5 to 10,000; and
wherein when n is greater than 1, the n Xxe2x88x92 groups can be the same or different.
According to another aspect of the invention, a microsphere further including an active agent is provided (see, e.g., Table 4). The microspheres into which the active agent can be loaded may include a complexing agent(s) to facilitate loading and/or modify the release of the active agent from the microsphere. Alternatively, the active agent can be loaded into the above-described microspheres which lack a complexing agent, e.g., the protein and/or the water soluble polymers of the invention can interact with the active agent to facilitate loading and/or modify its release from the microsphere. In general, although the active agent can be loaded into a microsphere of the invention during preparation of the microsphere, it is preferable to load the active agent into a preformed microsphere of the invention and, more preferable, to load the active agent into a preformed microsphere which contains a complexing agent(s) to facilitate loading and/or sustained release of the agent. In contrast to hydrogel microspheres, the microspheres of the invention do not swell significantly in water and, further, the microspheres do not require swelling in order to provide sustained release of the therapeutic protein and/or physiologically active agent from the microsphere.
As used herein, an active agent refers to an agent which has a diagnostic or therapeutic activity. Accordingly, an active agent can include a detectable label (e.g., a radioactive label) that is useful for identifying the locations of the released agent in vivo; Active agents also include therapeutic agents which are useful for treating a disease or condition. In certain embodiments, the preferred physiologically active agents are protein or peptide agents. Such protein or peptide agents typically can be further divided into categories, based upon the activity of the agent or the type of disease or condition that is being treated. The categories of physiologically active agents which can be used in the present invention include, but are not limited to, antibiotics, hematopoietics, antiinfective agents, antidementia agents, antiviral agents, antitumoral agents, antipyretics, analgesics, antiinflammatory agents, antiulcer agents, antiallergic agents, antidepressants, psychotropic agents, cardiotonics, antiarrythmic agents, vasodilators, antihypertensive agents such as hypotensive diuretics, antidiabetic agents, anticoagulants, cholesterol lowering agents, therapeutic agents for osteoporosis, hormones, vaccines and so on (see, e.g., Table 4).
The physiologically active peptide or protein which is employed in accordance with the present invention is a peptide composed of two or more amino acids. Preferably, such a peptide has a molecular weight greater than 200, e.g., in the range of about 200 to 200000. The more preferred molecular weight range is about 200 to 100000. More specific examples of physiologically active agents, including non-protein agents, which can be used in connection with the methods and compositions of the invention are provided in the detailed description of the invention.
According to still another aspect of the invention, a method for forming a microsphere is provided. The method involves: (1) forming an aqueous mixture containing a macromolecule, preferably a protein or a nucleic acid, and a water soluble polymer, preferably a carbohydrate-based polymer such as hetastarch; (2) allowing the microspheres to form in the aqueous mixture; (3) stabilizing the microspheres, preferably by contacting the microspheres with a crosslinking agent and/or exposing the microspheres to an energy source, preferably heat, under conditions sufficient to stabilize the microspheres; wherein the macromolecule is present in the aqueous mixture in an amount sufficient to form a microsphere that contains at least 40% and less than 100% by weight macromolecule. Although not wishing to be bound to any particular theory or mechanism, it is believed that the microspheres form during the mixing step; however, such initially formed microspheres are transient and require a further step (e.g., including a crosslinking agent in the mixture and/or by applying heat or other energy source) to stabilize the transiently-formed microspheres. Exemplary methods of preparing the microspheres are provided in the Examples.
According to yet another aspect of the invention, a pharmaceutical composition of matter and method of making said composition are provided. In certain embodiments, the composition includes a container containing a single dose of microspheres containing an active agent for treating a condition that is treatable by the sustained release of an active agent from the microspheres. The number of microspheres in the single dose is dependent upon the amount of active agent present in each microsphere and the period of time over which sustained release is desired. Preferably, the single dose is selected to achieve the sustained release of the active agent over a period of from about 1 to about 180 days, wherein the single dose of microspheres is selected to achieve the desired release profile for treating the condition.
According to another aspect of the invention, a syringe containing any of the microspheres disclosed herein is provided. For example, the composition can includes a syringe containing a single dose of microspheres containing an active agent for treating a condition that is treatable by the sustained release of the active agent from the microspheres. Preferably, a needle is attached to the syringe, wherein the needle has a bore size that is from 14 to 30 gauge.
Remarkably, the microspheres of the invention can be prepared to have a dimension which permits the delivery of microspheres using a needleless syringe (MediJector, Derata Corporation, Minneapolis, Minn. 55427), thereby eliminating the disposal problems inherent to needles which must be disposed as biohazard waste product. Thus, according to a particularly preferred aspect of the invention, a needleless syringe containing one or more doses of microspheres containing an active agent for treating a condition is provided. The microspheres can be prepared to have qualities suitable to be delivered by other parenteral and non-parenteral routes such as oral, buccal, intrathecal, nasal, pulmonary, transdermal, transmucosal and the like.
According to still other embodiments of the invention, nucleic acid-containing microspheres are provided. The nucleic acid-containing microspheres include: (1) a nucleic acid (e.g., plasmid, viral vector, oligonucleotide, RNA, antisense and missense nucleic acids); (2) a polycationic polymer (e.g., polylysine); and (3) a water soluble polymer (as described above). Thus, a method for forming the nucleic acid-containing microspheres is provided. The method involves: (1) combining, in one or more aqueous solutions, a nucleic acid, a polycationic polymer and a water soluble polymer to form an aqueous mixture which can be a mono- or multi-phase; and (2) subjecting the aqueous mixture to a crosslinking agent and/or an energy source under conditions (e.g., of concentration, incubation time) sufficient to stabilize a microsphere. Exemplary methods of forming the nucleic acid microspheres are provided in the Examples.
These and other aspects of the invention will be described in greater detail below. Throughout this disclosure, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains unless defined otherwise.